摘要
目的探讨死亡相关蛋白激酶(DAPK)与肿瘤抑制基因p73基因启动子区CpG岛在不同类型白血病细胞基因组中存在特异甲基化位点的情况。方法通过亚硫酸氢盐测序法,研究DAPK与p73基因启动子区CpG岛在单核细胞系白血病细胞株U937、淋巴细胞系Jurkat、粒细胞系HL-60和正常人白细胞基因组中的甲基化状态,收集测序结果,并对不同CpG位点甲基化率进行计算;对上述4种细胞来源基因组中的DAPK与p73基因CpG岛甲基化模式图进行绘制,并对其特异甲基化位点组合进行筛选。在40例正常对照、82例白血病患者外周血标本和白血病细胞株中,采用甲基化特异性PCR法对基因异常甲基化模式的诊断价值进行验证。结果测序含有亚硫酸氢盐测序法产物转化质粒的菌株共106个,且成功绘制DAPK与p73基因CpG岛甲基化模式图。白血病细胞株DAPK与p73基因去甲基化程度较正常细胞基因组明显升高。在白血病粒细胞系HL-60中,DAPK基因与其它白血病细胞株甲基化状态明显不同。在白血病细胞株和正常细胞中,p73基因甲基化状态完全相反。DAPK基因甲基化检测可有效鉴别粒细胞系与淋巴细胞系白血病,其对急性非淋巴细胞白血病临床诊断的敏感度为68.57%(24/35),特异度为100.00%(27/27),准确性为82.26%(51/62);p73基因甲基化检测可有效鉴别白血病细胞与正常细胞,其对白血病的临床诊断敏感度为29.03%(18/62),特异度为100.00%(40/40),准确性为56.86%(58/102)。结论在不同临床分型的白血病细胞基因组中,DAPK与p73基因启动子区CpG岛具有特异性的甲基化位点,可用于初步评估白血病不同分型,因此可作为临床重要的潜在肿瘤标志物,能够为后期探究白血病肿瘤甲基化标志物提供可行的检测方法和良好的实验基础。
Objective To investigate the presence of specific methylation sites in different types of leukemia cell genomes in CpG islands of promoter region of death-associated protein kinase(DAPK)and tumor suppressor gene p73.Methods The methylation status of CpG islands in promoter region of DAPK and p73 gene in monocyte leukemia cell line U937,lymphocyte line Jurkat,granulocyte line HL-60 and normal human leukocyte genome were studied by bisulfite sequencing.The methylation rates of different CpG sites were calculated.The island methylation pattern map was drawn and the specific methylation site combinations were screened.Methylation-specific PCR was used to validate the diagnostic value of abnormal gene methylation patterns in peripheral blood samples from 40 normal controls,82 leukemia patients and leukemia cell lines.Results 106 strains containing transformed plasmids produced by bisulfite sequencing were sequenced in this study,and the methylation patterns of DAPK and p73 CpG islands were successfully mapped.The demethylation of DAPK and p73 genes in leukemia cell lines was significantly higher than that in normal cell genomes.In leukemia granulocyte line HL-60,the methylation status of DAPK gene is significantly different from that of other leukemia cell lines.In leukemia cell lines and normal cells,the methylation status of p73 gene is completely opposite.DAPK gene methylation detection can effectively differentiate between granulocyte and lymphocyte leukemia.The sensitivity,specificity and accuracy of DAPK gene methylation detection for clinical diagnosis of acute non-lymphocyte leukemia are 68.57%(24/35),100.00%(27/27)and 82.26%(51/62),respectively.p73 gene methylation detection can effectively differentiate leukemia cells from normal cells,and its sensitivity for clinical diagnosis of leukemia was 29.03%(18/62),specificity was 100.00%(40/40),accuracy was 56.86%(58/102).Conclusion The CpG islands in the promoter region of DAPK and p73 genes have specific methylation sites in the genomes of leukemia cells of different clinical types,which can be used to preliminarily evaluate different types of leukemia.Therefore,DAPK and p73 genes can be used as important potential clinical tumor markers and provide a feasible detection method and a good experimental basis for exploring the methylation markers of leukemia tumors in the later stage.
作者
王浩
杨瑛
梁华
WANG Hao;YANG Ying;LIANG Hua(Department of Endocrinology and Hematology,The 942 Hospital of PLA,Yinchuan Ningxia 750004,China;Department of Anesthesia and Surgery,The 942 Hospital of PLA,Yinchuan Ningxia 750004,China)
出处
《临床和实验医学杂志》
2019年第19期2085-2088,共4页
Journal of Clinical and Experimental Medicine
基金
宁夏科技支撑计划项目(编号:20112YS274)
